tRNA, or transfer RNA, is an adaptor molecule that serves as the physical link between mRNA and the peptide sequence of proteins. It plays a vital role in transferring amino acids to the growing peptide sequence during the translation process from mRNA to protein. Typically, it ranges from 76 to 90 nucleotides in length and has a secondary structure resembling a cloverleaf shape, with a tertiary structure forming an “L” shape that enables it to fit into the P and A binding sites of ribosomes. The tertiary structure is formed from the secondary structure through coaxial stacking of helices, a common feature in RNA molecules. Figure 1 depicts the secondary and tertiary structure of tRNA.
Figure 2 illustrates an example of the secondary structure of tRNA with all the nucleotides labeled. The tRNA secondary structure consists of three hairpin loops, giving it a cloverleaf shape. The main constituents of tRNA include:
- The acceptor stem – Composed of 7-9 base pairs, the acceptor stem forms by pairing the end of the 5′-terminal nucleotides and the end of the 3′-terminal nucleotides, with the 3′ CCA tail excluded and overhung from the acceptor stem. The base pairing of the stem can be either Watson-Crick or non-Watson-Crick.
- The 3′ CCA tail – Overhung from the 3′ end of the acceptor stem, the CCA tail comprises a cytosine-cytosine-adenine sequence. Amino acids load onto the tRNA through covalent binding with the 3′-hydroxyl group in the terminal adenine nucleotide. This CCA sequence is critical for the recognition of tRNA by enzymes and plays a crucial role in translation.
- The D arm – This hairpin structure contains a D loop at the end and includes modified uridine, dihydrouridine (D), as highlighted in Figure 2. The stem of the D arm can vary in size and sequence among different tRNAs but typically consists of 4-6 base pairs. The D arm contributes to the stability of the tRNA structure.
- The anticodon arm – Similar to the D arm, the anticodon arm is a hairpin structure with an anticodon loop at the end. The anticodon stem has 5 base pairs. The anticodon loop, a crucial element of tRNA, contains a three-amino-acid sequence (or “anticodon”) in reverse order that can form base pairs with the complementary mRNA sequence during translation. This step ensures that the correct amino acid is added to the growing polypeptide sequence to form the correct protein.
- The variable arm – Situated between the anticodon arm and the TΨC arm, the variable arm varies in size and sequence among different tRNAs. The number of nucleotides ranges from 3 to 21 bases, contributing to the stability of the tRNA molecule.
- The TΨC arm – This hairpin structure has a TΨC loop at the end, containing modified thymidine (T) and pseudouridine (Ψ), which gives the arm its name. These modifications contribute to the stability and correct folding of the tRNA.
A significant challenge in applying tRNA in therapeutic areas lies in the large number of modified nucleotides, often resulting from methylation or deamination. These modifications can sometimes affect the tRNA’s interaction with ribosomes and may occur in the anticodon, altering base-pairing properties. We still have a very limited understanding of tRNA molecules as a whole because these modifications are challenging to analyze, and the molecules are also difficult to chemically synthesize. As our experience with tRNA molecules deepens, we will become more capable of utilizing these unique molecules to address the unmet needs of patients.
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